Interference microscope



Patented Dec. 1, 1953 UNITED STAT *ATENT OFFICE Claimspriority; application Great Britain; June 7, 1948' Claims. 1;

This invention? relates to? inioroscorresi' and has for an: object to provider an improved interference microscope in which? a :high numerical aperture oi:both theiviewingtand'ailluaninatirig'systems can? be attained.

An interference microscope is optical system-ibr-examiningan object iii such a way-thatlocalx variations in phase retardation introduced by the? object" (occasioned: by' differences in refractive: index! or in thickness oil thematerial: or in he'ight-ofi its reflecting surface) become-apparent as differences in light: intensity orcolour. It. can therefore-beused inter-aha; for examining colourless objects such as: living: cells;

A well-known: example of: interference microscopy is theso-ca-lled phase-contrast: method: of Zernicke (Physica 9 SZKSAWQ, 194(2). in which light scat-ter'edby'the object is caused to interfere' withlight directlytransmit-ted by it.- An other example: is: theuseof: a partially'reflectingslide and cover-slip (lVier-ton; Pros. Roy Soc. A; 189;, 309 and 191,1: (1947-) also Ambrose Journ; Sci. Instr. 25-, 137* (1948)): where light which has passed directly through the: system interferes with light-which has been multiply reflected by the-slide. and cover-slip. The Zer'nicke method has:- the drawback that any scatteredlight coincidingv-in-idirectioirwith direct light has thewrengphase and acts-against the'remaining scattered light. In the Merton method! only a: narrow beamof light may.- be used unless. azone plate (which is difficult to make for operation. at more than one wavelength) is'inserted This-is. bee cause the retardation otthe. rays in a beam of light whichhas beenirefiected by. the two partial reflectors dependson the angle which they make with the optic axis.

The'inventicn may in one aspect be defined as a microscope comprising" an' interferometer in which abeam of'liglit' is-split by partial reflection into two parts diverging from each other, the parts being recombined to show interference effects; and an objective lens system common to both par-ts oi he sp'lit-bearn; so that-interfering (ii 6. vectoriallycombining) imageso ian object plane in: the: objective:- lens system are: formed with locally.- difiering; nhasecretardationp The split: beams whichi interfere with one anothenmay becaused to? throughzthe objecttive lens? system either in oppositehalyes of the 2. field or in opposite directions round an angular. loop path.

In a simple" optical system or the invention wherein the split beams pass" in" opposite dimetions round an'angular p path; these beams; though passing through the same objective, do not coincide but pass through on oppositesides'of the axis of the objective; Co-inciden'ce is secured, according to a further'featur'ei of the; in-.- vention by providingfor a relative rotation of the split beams'through'180 about the axis; For the beam rotation either: a" 16115.01 a'prismsystem' may be employed;

Other features"oftlieinvention are set out in the appended claims;

In" carrying out 'theJinVentiQn; partial reflectors for splitting and reconstituting the beam are obliquely inclined to the incident lig t rays,'. as in the'well known Michelson interferometer, so that the split beamsbranchout on'separate'axes and arerecornbiheditofall o'n commonaxes. This facilitates the formation ofco' incidentimages whereas in: the Merton system the images fall one 'behind'the other as a: consequence of the development" of? co -axial beams from partialreflectors normaltb and spacedalong the-axis.

Furthermore the partial t reflectors.- arelocated not in the region of the object plane as" in the Merton systemibut" atpoints remote from such planes; where, even high aperture con.- dens'ers and objectives,- the light rays make only smallangleswith their optic axis.-

The essential functional parts; of an: interference-microscopef-can be" enumerated asfollows; itibeing understood that in'praetice. several func' tionscan be performed by" a common element A'firstpartial reflector splitslight from asource into two similar beams; The beams each pass through objectives to "the secondpartial reflector; where they: are recombined to formtwo new beams whichmaybe termed the odd beam and the" even beam;

The: even beam: contains light which. has; been transmitted by, both? partial reflectors combined with light which. has been reflectedrby botln partial reflectors; The odd beam contains the remaining light of the split beams; namely that transmittedby't-he'first partial reflector and re"- flec'md by thei secondicombinedwith that reflected by the first and transmitted by the second. The

odd and even beams are complementary in the sense that a diminution of light by interference in one of them is compensated by an increase by re-inforcement in the other.

The even beam and the odd beam each contain two superposed images formed by the objectives in the split beams. If an object be placed in front of one of the objectives, the retardation of the light due to the refractivity of the object causes the interference between the two images to differ from point to point so that the object is seen in the form or a contour-map of phase retardations.

The split beam having no object in it constitutes with the original beam or in some other direction. 2

Actually some of it, particularly from very small objects, will travel backwards, and in some forms of the invention the backward scattered light is collected and, may or may not be used to improve the visibility of the object. Also in some forms a beam may pass more than once through the object, producing scattered light in each transit.

In practice, the microscope will usually inelude condensers in the split beams to illuminate the object planes at high aperture, a collimator to permit of the use of a small source. and an eyepiece comprising a field lens to collect light from the objectives and an eye lens to magnify the image further as is usual in microscopy. The term lens is used throughout as an abbreviation for lens or systems of lenses or curved reflector or system of curved reflectors.

The invention will be further described with reference to the accompanying drawings in which: Fig. 1 is a known optical svstem from which the invention is developed. Fig. 2 shows a variant due to Linnik and described by W. Von Kinder in Zeiss Nahrichten, August, 1937 and Figs. 3 to 13 show microscope systems embodying the invention and detailed variants therein.

In the arrangement of Fig. 1 light from a source S is projected by a collimator El upon an obliouely-inclined partial reflector R! consisting for example of two prisms cemented together with a film of aluminium, silver or rhodium deposited frcm vapour between them. The light transmitted by the partial reflector passes through a condensing lens OI, an object plane Pi, and an objective lens 02, and is then reflected at a mirror R3 on to a second partial reflector R2 similar to RI. Thence the reflected part of the light forms part of the odd beam described above and passes to an eyepiece E2 comprisin a field lens and an eye lens. so that an image of the object plane is formed at I; the transmitted part of the light forms part of the even beam and. is rejected at R2. The light reflected by the partial reflector R! is reflected at a mirror R4 and then passes through a condensing lens 03, an object P and an objective lens 04 to the second partial refiector R2. Thence the transmitted light enters the odd beam and forms an image of the object at I; the reflected light is rejected in the even beam.

. the odd beam to an eye-piece E2.

' reflected to the eye-piece.

If no object is in position and the media at the two object planes are similar, the system may be adjusted so that the odd beam is dark because its components are in phase opposition. Then introduction of the object modifies the phase of the associated component beam and the object is seen as an image of brightness or colour dependent on the variation in refractive power from point to point in the object.

This optical system of Fig. 1 is easy to free from stray light due to unwanted reflections because the light is unidirectional through the branches of the system; but for the same reason the light passes but once through the object and the backwardly scattered light is lost so that image brightness is lost. Fig. 1 may be regarded as a form of the well-known Jamin interferom" eter' with a microscope and its object introduced into one branch and another microscope, or the equivalent, Without an object, introduced into the other branch. The Linnik system of Fig. 2 and the systems of the invention may be regarded as applications of the Michelson interferometer.

In the prior Linnilc system in Fig. 2. a single partial reflector serves for splitting the beam and for recombining the split parts which are reflected to it by mirrors R5, R5. Light from the source S and collimator El is split at the partial reflector RI, the reflected light being condensed in a lens Oi reflected at a mirror R5 to return through the lens OI, which now serves as an objective lens, to the partial reflector whence part is reflected into the even beam which returns to the source and part is transmitted The light from the collimator which is transmitted at the partial reflector follows a similar path through a lens 03, returning from a mirror R6 to be partially transmitted to the source and partially Thus interferrine: images of an object P and of an object plane 39% are formed in the eye-piece.

The system of Fig. 2 differs from that of Fig. i in that light passes twice through the object and the backward-scattered light is collected;

but there are two image planes (direct and reflected) which do not coincide unless the object coincides with the mirror B6. Therefore, except with very thin objects, half the scattered light is out of focus and is merely a nuisance. Furthermore, the path of the backward-scattered light is shorter or longer than that of the for ward-scattered by twice the object-mirror dis tance so that some of it will have the wrong phase except in special cases.

Fig. 3 shows a simple and effective form oi interference microscope which can be made from an ordinary one merely by insertion of a special eye-piece. It is similar to the arrangement of Fig. 2 as regards beam paths, scattered light etc., but instead of two objectives and the cornplication in adjustment thereby involved, it uses two halves of a single objective. The field of view is divided into two semi-circular fields which are superposed and made to interfere. As shown. llght from source S is reflected by a prism B": through an eye-piece field lens L and is then refracted in a double prism K towards a pantially reflecting surface R! in the prism. The reflected part and the transmitted part then follow the paths shown through lens 0!, object plane P and mirror R5. Replacement of the mirror by a lens 05 and a spherical mirror Rt as shown in Fig. 4.- allows the reflected image to 5 be brou'g-lit'"- intocoincidencewith the direct image.

It will be appreciatedthatiif thearra'ng'ement oi l ig 3 01' Fig; 4 one half-of the obj'ect is com pared with the other half l Such an arrange" ment is suitable for examination oi? thin suspensions of: bacteria, etc: where the super posi tio'n of' twohalf-fields causes no confusion.

In Fig. Sis-shown a prism 5' with: apartia'll'y reflecting interior siirface "I a collimator El and an eye-piece as which can he: used" instead of 'th'e elements RLRI, 13. K; ofFig. s or Fi'gi 4 Its surfaces are used as rene cti fleeting surfaces I to avoid chr Anf embodiment of theinvention: invoivin'g; an angular loo-g the beam path". isf'shuwn: in Frgt. 6. Light fr'Ofnr6231 '1 vil filia'l SOUI'GPJ i- {5063' throughacoilimator ji I thence viathe semi reflector R and the full refiectors?HS,.l3zlilte-tlie objectives ()8, O9 travelling in both directions round the: closed circuits; so than each. objective acts a condenser for: one: direction. and; as an objective for the: other' direction. 'l he lig-ht then emer es fl om: the-system as anoddi bean'i returning to source S an everr beamentering: the eye orc'ainera through? eyepiece E21 ln the-a b; sense of an ohject th partition of returning light between El and E2 depends on the align ment of the objectives and on the: position-' of the planeof Bi relative to'the lin'e' of: intersection or" R9 and Bit; Adjustment of this peertion can beinade tocompensate for small: errors inalignment; thus? optimal interference can be secured merely by removing: the: eye-piece and adjusting the tilt oi. a' mirror" (e. mirror Rzi'e about two axes until thewhole aperturev of; the objectives is seen-to be. filledby' one interference fringe (necessarily the central one-).. If: now an object" l? is introduced the phase. of: both beams is altered to an equal extentihy' passagethrough the object; so that they are still: in phase'opposie tion regards enteringEl): provided: they remain suitably superposed; Ererrifz the obiect-is markedly inhomogenous in. refractive index: or thickness inits' two images formed in: E2 at liby the objectives? O8; O9 respectivelywill. continue to be opposed like the illuminating beams; rovided that they overlap suiifici'ently accurateln How-- ever, if the overlapof the: two images; is? destroyed by deiocussing one of them,.thei :interference will be destroyed, and anysmallregion where the phase change diiiersbyother than a whole number of wave-lengths f-rom the average over the area occupied by its deiocussedl image will appear bright? on dark background. If a small object containsstill smallersub-objects' the defocusing can be so adjusted that the object. is dark like. the background except at its edge while the sub-objects are bright.

An alternative-'- way of making the images in Fig. 6 diiien. in their. phase rotation, from the background is basedon: the phase change:-whichv light undergoes on passing througha: focus; If. one image is formedslightly in front of the focal plane. of. the. eye-piece and ther other image: is

formed. slightly behind that focal. plane; they should combineto formsa'bright image onadark" background. This. image will" be. but slightly out" of focus, because the. distance Within which. the phase-change occurs ati a focus issaid to be-of: the order of a wavelength.

In Fig; 6, the backward-scatteredlight? is collected. It is'in phase with the forwardl-scattered light if the optical: path between: the: semi:- reflector and the obj set is equal. in: the two directi'ons; Whemthi 'conditioniissatisfi'edu the2backwall-scattered? light: from one: beam can. exactly replace that from the other beam which has gone round the: wrongyway andihas' thereby been defocussed; Thus, with. an objective. having a numerical. aperture equal to that of. the. medium containing thembJ'ectg-the focussed image is in effect! formed. from light collected over awhole sphere, instead 05;. overs: hemisphere; This may give: anunusually: high. resolving; power, but the theory of suchi a 'caseis not. yet properly understood. Apartv from: this possibility; the mainadvantage'of Fig'; firis: inisimplicity and ease of. adjustment. A slight disadvantage is that the system: uses? the? even beam; notithe: odclhealm, so thatinterfer'enceis:complete only if Rl is exactly semi neflecting; for it b is the fraction of light reflected; and l hthat transmitted, the two beams whichare'requiredto interfere haveintensiti'es? b and i b) instead: of equal intensities hil h) as-in'. the'other examples which usethe odd: beam. To obviate the need for an exact semi-reflector; the odd. beam may be used: with the aidofia secondpartialrefiector RI I as shown in 7. Theses-end partial reflector leads to some'vvastage of light; it enables a black background to be achieved with an. inexact semirefiector but; for the. spherical aperture effect mentioned above coho-perfect truesemi-refiection isstill necessary Figst 6 and. E represent a cyclic type of interferoineterv in that light travelling down each of the interferometer reappears travelling up the other arm. Thisv closed-circuit principle was used by Michelson and Morley- Lodge for studying ether. driftand by Zeeman for studying phase-change on reflection but its valuable properties. in= relation. to. microscopy have not previously been recognised. or applied..

The arrangement of Fig. 3 can be modifiedto a-eyclic type; by? replacingfthe by two right-angled prisms. R12, R1 cemented to the objectiyeQL asshowniin Fig- 8;, this. variant is suitable forusawith low. power objectives where the restricted size'of. the object. is no disadvain tage..-

Instead of theispacedrenecting elements Ri, Rai Rlfl on Fig... 6 arrangements. of such as-showninlilig. 9 oz: Fig. lil may be used.

The exampleshown.inhig. 3-- can be used for examining reflecting objects, which can. replace themir-ror R5;- sma'llrlocal diiierences in height of. thesurface of the: object then cause differences in=inter-ference;-- An important. application is to metallurgy-o The microscope optical systems described may be set: up in instruments using techniques well known-in microscopy.-

Figs. 1-1 and. 12 are two. sectional. and. partly diagnammatic'views of a-inicroscope. having novel constructional features and embodying the. optical systems-of. Fig; 6.-.

The; instrumcnticf. Figs. 1 L and 12 comp-rises a frame- 2-0, ii-shaped in plan; and having three legs: 21:, 22, 23... As. shown, the frame: con :ises am optical bench. 24 on. which are mounted a lights-source 25 and. collimatingz or beameforining lenses: 2.5;. 211.. Oneand support. for the optical bench is provided by a housing comprising a. platformfi-lflwalls iii and a top-plate as.

A reflecting prism {it reflects the beam from the-optica1 bench towards an inclined reflector 32 attached to the. top plate. Light incident on this reflector is directed towards a similar'in clinedi reflector. 33* mounted on the platform, and

thence towards an eye-piece 3 5 adjustable in a barrel fixed to the wells 28 by means of a bracket 36.

Brackets 33's and 38 project inwardly from the walls and standards sc rise from the platform to provide adjustable three-point supports for two L-shaped sub-frames 49, M.

Sub-frame has mounted upon it a partial reflector unit 52 comprising two prisms with a partial reflecting surface between them. Subfrarne fl! has mounted upon it two accurately aligned ob'ectives and provision is made for the insertion of slides between these objectives. A pivoted stage (not shown) for the slides may be arranged to move angularly in a horizontal plane to carry the slides into position.

A camera be arranged above the optical bench on a parallel axis to receive light from a reflecting prism as (similar to the prism 3|) located in the light path to the eye-piece 3i. Prism it may be removable to provide for direct viewing or photogrephic recording as alternatives; or it may be partially reflecting to permit direct viewing for iiustrnent and timing purposes concurrently with the taking of photographs.

From the foregoing description of the instrument with reference to Figs. 11 and 12 it will readily be seen that the optical system is that of 6, with a reflector inserted between the light collimator the partial reflector for convenience in disposing the components, and with [1'1 optional reflector in the outgoing light path for use in photographic recording.

It will be seen also that the objectives on their sub-frame it are readily removable and replaceable, and that the partial reflector on its subframe is likewise removable. In the absence of the partial reflector the instrument functions as an ordinary microscope without interference effects.

In the optical arrangement of Fig. 6 the interfering rays, though going through the same objective system do not coincide but pass through on opposite sides of the objective axis. This non co-incidence can be avoided by a relative rotation of the beams through 130 about the optic axis. All interfering rays traversing the centre of the fleld then coincide everywhere, while for other parts of the field they lie parallel and close together. The result is that even bad optical components can give a dark interference spot in the centre of the field, darker than even high-quality components give with unrotated beams. The beanie may be rotated by either a prism or lens. A lens is advantageous in that it can i l y a condenser-formed image to the same size objective-formed image, so that cyclic interference microscope with reversing lens can one objective and a good condenser instead of the two objectives which are needed without the reversing lens. This not only reduces the cost but enables the object to be mounted between a slide and coverslip in the ordinary wey instead of between two coverslips as is required by the two objectives. Such an optical system can be incorporated in a standard microscope with but small alteration. The reversing lens provides a further advantage in that it causes the interference fringes to be focussed in the same plane as the object instead of near the back lens of the objective. This allows the object to be seen as either white on black or black on white, merely by moving it about the field.

This further form of the invention is exemplifled in, and will be more fully understood, by reference to the optical system of Fig. 13. Light from a source S and a collimator E1 is directed upon a partial reflector RI. The transmitted part of the beam passes to a mirror RIG thence to a mirror R9 and back to the partial reflector where it is partly transmitted to an eye-piece E2. The reflected part of the initial beam traverses the same angular loop in the reverse direction and is partially reflected to the eye-piece E2.

The system diifers from that of Fig. 6 in that a reversing lens U3 and a fleld lens Ll l cooperating therewith, are inserted in the angular loop. This permits the use of a, conventional objective 013 and co-operating condenser OM with the object plane P l 3 between them. Normal slides and cover-slips can be inserted at the object plane. Images of the object are formed at PM and P15.

I claim:

1. An interference microscope for the examination of objects through which light can pass, comprising, in combination, a collimator, a partially reflecting, partially transmitting reflector disposed in the path of a light berm from the collimator with its partially reflecting, partially transmitting surface set obliquely to this path, two reflecting devices disposed one in the path of each of the reflected and the transmitted part beams from the reflector at angles such that the part beams are redirected to travel in opposite directions along one and the same path between the reflecting devices, means for supporting an object in, and in a plane transverse to, the common path of the two part beams, two objective lenses disposed one on each side of the object plane and acting each as a condenser for light traveling in one direction and as an objective for light traveling in the other direction, and an eyepiece disposed in the path of a composite image-forming beam derived from the two beams passing away from the object plane along the common path of the two original part beams toward the reflector, the power and positioning of each of the objective lenses being such that the two images which are to interfere are formed as images of equal size lying in at least substantially the same plane at an appropriate distance along the light path to the eyepiece.

2. An interference microscope as claimed in claim 1, having a second partially reflecting, partially transmitting reflector situated in the light path between the first mentioned reflector and the collimator in position to reflect to the eyepiece a part of the composite image-forming beam which it receives from the first mentioned flector.

3. An interference microscope as claimed in claim 1, having a frame, a three-point mounting for the frame, and a third reflecting device, the collimator being mounted on the frame and extending in a vertical plane containing two of the mounting points, the reflector, reflecting devices, objective lenses, and eyepiece being dispose: in the vertical plane containing the third mounting point and one of the two first mentioned mounting points, the third reflecting device being positioned for reflecting the beam from the collimator to the reflector, and supporting means for the members disposed in the second mentioned vertical plane.

l. An interference microscope as claimed in claim 3, including detachable subframes for the reflector and for the objective lenses.

5. An interference microscope as claimed in 9 10 claim 1, further comprising a reversing lens and References Cited in the file of this patent a cooperating field lens interposed in one of the UNITED STATES PATENTS two part-beam path sections between the partial reflector and the objective/object-plane/objec- Number Name Date tive assemblage, the power and positioning of the 5 1570,77) s1dentpf 2 last named lenses and of the two objective lenses 1,573,401 Heme 1926 being such that the two images which are to 2365,78 Von Baeyer 1941 interfezkelanctll bebfgrnled astilmages of 1equal size F REIGN PATENTS ie in a eas su s an ially e same p ane at an appropriate distance along the light path to the w gg g gggg Oct 55 1911 eyeplece- 355,911 Great Britain Sept. 3, 1931 474,809 Great Britain Nov. 8, 1937 JOHN LEGER PHILPOT- 555,672 Great Britain Sept. 2, 1943 

